Geographic atrophy (GA), an advanced form of age-related macular degeneration (AMD), is a major global cause of blindness that affects more than 1 million Americans. Of the many pathological features that comprise GA, one of the cardinal hallmarks is degeneration of the retinal pigmented epithelium (RPE), which also is an FDA- accepted endpoint for registration clinical trials. GA remains an unmet medical need because, in large part, the mechanisms that promote RPE degeneration are not fully resolved. Earlier, we identified a specific accumulation of toxic non-coding Alu RNAs in the RPE of GA eyes that results from a deficiency in DICER1 (Nature 2011), which triggers RPE degeneration by activating the canonical caspase-1 inflammasome (Cell 2012). Recently, we elucidated the signaling symphony that orchestrates this cellular toxicity: disruption of DICER1:Alu RNA homeostatic regulation induces non-canonical inflammasome activation, a molecular cascade mediated by caspase-4/11 and a novel, non-lytic gasdermin D (GSDMD)- dependent activation of caspase-1 and secretion of IL-18 (Nature Medicine 2018). Enhanced levels of these molecules were also identified in human GA, marking it as the first non-infectious human disease associated with the non-canonical inflammasome. Combined with our exciting preliminary data that amyloid ?, another trigger of RPE degeneration, also activates the non-canonical inflammasome, these findings suggest that the non-canonical inflammasome could be an integrator of multiple toxic signals that drive RPE degeneration in GA. Given that the inflammasome responds to myriad triggers of cellular distress, it could represent a critical checkpoint that triggers cell death, and therefore an attractive target for halting RPE degeneration. However, we still lack an integrated understanding of inflammasome activation in GA, and how it induces RPE degeneration. A rigorous definition of these mechanisms is crucial to enhancing our understanding of the molecular drivers of this hallmark of GA and to developing rational treatments. We will provide novel functional insights into how dysregulated non-canonical inflammasome activation contributes to RPE degeneration via the following thematically integrated yet independent Aims: (1) Create a spatial map of the non-canonical inflammasome pathway in human donor eyes; (2) Define the role of Gasdermin D (GSDMD) in non-canonical inflammasome activation; (3) Determine whether targeting the non-canonical inflammasome pathway ameliorates RPE degeneration in acute and chronic animal models. These studies will illuminate novel aspects of the molecular and biochemical bases of RPE degeneration, and help validate a molecular targeting strategy that could be translated into clinical trials. As such, this proposal is aligned with the 5-year goals of the NEI's Retinal Diseases Program strategic plan.